All blood cell types that make up the adult cardiovascular system are created and maintained by self-renewing, multipotent hematopoietic stem/progenitor cells (HSPCs). Conserved across vertebrates, HSPCs are specified early in development, arising from a unique population of vascular cells called the hemogenic endothelium. Given the difficulties in the ex vivo differentiation and expansion of HSPCs from pluripotent stem cells for regenerative therapies, it is readily apparent that knowledge about HSPC specification is significantly lacking. This gap in understanding stems from the challenges in visualizing HSPC emergence in mammalian embryos that develop internally. The proposed study will address these biological problems with the zebrafish model because the transparency and external fertilization of zebrafish embryos will allow for the molecular dissection of the mechanisms that control this dynamic endothelial to hematopoietic transition (EHT) in vivo. Small, non-coding microRNAs (miRNAs) are key regulators of stem cell and cardiovascular biology because of their ability to post-transcriptionally silence a diverse array of target genes. While miRNA regulation is crucial for HSPC self-renewal, maintenance, and differentiation into multiple lineages, the role for miRNAs in hemogenic endothelial specification and HSC emergence is largely unexplored. From a reverse genetic screen to identify the cardiovascular function of endothelial-enriched miRNAs in zebrafish, mir-223 was identified as a novel candidate regulator of HSPC specification because the loss of mir-223 led to an expansion of HSPCs at the onset of HSPC induction. Thus, the goal of this proposal is to directly test the hypothesis that mir-223 functions to negatively regulate the transition from endothelial to hematopoietic cell fates. By employing an innovative strategy to create a mir-223 fluorescent reporter, mir-223 localization patterns will be assessed for the first time in vivo and correlated with the dynamic emergence of HSPCs from the hemogenic endothelium in zebrafish. The proposed research will also examine whether mir-223 functions in HSPC production by visualizing changes in hemogenic endothelial specification and cellular behaviors of emerging HSPCs when mir-223 activity is lost or elevated. Finally, the genes that are directly targeted by mir-223 during EHT will be identified by their upregulation in mir-223 mutants compared to wildtype hemogenic endothelial cells. Altogether, this proposed work will implicate miRNA regulation as a fundamental mechanism of HSPC specification, and will better define the genetic network regulating this process. Importantly, these studies will establish the zebrafish mir-223 mutant as a new leukemogenesis animal model, and will have the potential to instruct regenerative medicine approaches in the ex vivo production of HSPCs for the treatment of leukemia and other blood disorders.